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Actions of Ya-hom, a herbal drug combination, on isolated rat aortic ring and atrial contractions
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Phytomedicine 12 (2005) 561–569 www.elsevier.de/phymed
Actions of Ya-hom, a herbal drug combination, on isolated rat aortic ring and atrial contractions W. Suvitayavata,, S. Tunlerta, S.S. Thirawarapana, C. Kitpatia, N. Bunyapraphatsarab a
Department of Physiology, Faculty of Pharmacy, Mahidol University, 447 Sri-ayuthaya Road, Bangkok 10400, Thailand Department of Phamacognosy, Faculty of Pharmacy, Mahidol University, 447 Sri-ayuthaya Road, Bangkok 10400, Thailand
b
Received 22 January 2004; accepted 5 March 2004
Abstract The effect of the Thai popular medicine Ya-hom on cardiovascular function was studied in isolated rat aortic ring and atrium by comparison with norepinephrine (NE). Water extraction of Ya-hom at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml stimulated aortic ring contraction dose-dependently. The maximum contraction, at 16.67 mg/ml, was about 14% that of NE. This stimulatory effect of Ya-hom was inhibited partially by phentolamine, which indicated that the effect of Ya-hom was partially dependent on the a receptor, similar to NE. Administration of Ya-hom with NR decreased the force of aortic ring contraction as compared to the effect of NE alone, indicating that Ya-hom may have a partial a-agonist activity. Ya-hom at concentrations of 1.67, 8.33 and 16.67 mg/ml showed a dose-dependent, positive inotropic and negative chronotropic effects. Ya-hom increased the force of isolated atrial contraction with a slow onset and prolonged action. In contrast to norephinephrine, which acted on b1 receptor, causing positive inotropic and chronotropic effects, propranolol did not alter the effect of Ya-hom on the atrial contraction. This shows that the action of Ya-hom on atrial contraction does not involve b receptor. This study demonstrated that the selected Ya-hom preparation increased vascular smooth muscle contraction, and increased the force but decreased the rate of atrial contraction. r 2005 Elsevier GmbH. All rights reserved. Keywords: Ya-hom; Aortic ring; Rat atrium; Cardiovascular effects
Introduction Ya-hom, one of the most popular Thai folk medicines, has been used for treatment of fainting, nausea and vomiting. Ya-hom preparations are marketed under different trade names with different compositions as well as different proportion of medicinal plants. The Corresponding author.
E-mail address: [email protected] (W. Suvitayavat). 0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.03.014
cardiovascular effects of some principal ingredients in Ya-hom have been reported. Aquilaria agallocha Roxb (Benbassat et al., 1959), Kote-sor (Khadzhai and Sokolova, 1960) and Chinese cinnamon (Kong et al., 1976) have been reported to cause vasodilation and decrease blood pressure, whereas aqueous and alcoholic extracts of Kote-kra-dook caused an increase in blood pressure (Bose et al., 1961). The methanol extract of Kote-kra-dook, however, inhibited KCl-induced aortic contraction (Shoji et al., 1986) and was also shown to
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have a hypotensive action (Gupta and Ghatak, 1967). Reports showed that licorice decreased (El-Mahdy et al., 1973; Shihata and Elghamry, 1963) and increased blood pressure (Hano et al., 1950; Fournier and Lagrue, 1971; Girerd et al., 1958), stimulated and depressed myocardium function and caused vasoconstriction and vasodilation according to concentration (El-Mahdy et al., 1973). Most of the Ya-hom recipes are composed of both cardiovascular stimulatory and inhibitory herbal medicines. Therefore, the exact action of each formula must be investigated. There have been few reports about the effect of Yahom on both animal and human cardiovascular function. Ya-hom slowed the pulse rate and widened the pulse pressure in humans (Matangkasombat, 1973). In the rat, the water-soluble fraction of a chloroform extract and a water extract of Ya-hom extracts raised blood pressure, whereas the alcohol extract and waterinsoluble fraction of the chloroform extract lowered blood pressure (Matangkasombat, 1974). In addition, Ya-hom extracts have a direct stimulating effect on the rat heart, whereas they show no effect on the rabbit heart (Matangkasombat, 1974). Another report using water extraction indicated that four brands of Ya-hom decreased blood pressure but had no effect on the heart rate, pulse pressure or electrocardiogram of rats (Wangmad et al., 1986). Two of these four brands (nos. 1 and 4) had a positive inotropic effect, two of them (nos. 1 and 3) had negative chronotropic effect, whereas the remaining one (no. 2) had no effect on the isolated atrium (Na Pattaloong and Sawasdimongkol, 1995). According to some controversial results, determination of the effect of Ya-hom on the cardiovascular function remains inconclusive. This may be due to use of the different brands, extraction methods and experimental models. Because most Ya-hom formulae contain similar major ingredients, the most famous brand of Yahom has been selected for the study of its cardiac and vascular effects, in vitro in this present study and using an in vivo model for the next study. The pharmacological actions of this brand have never been reported. A massive single preparation of a lyophilized water extract of Ya-hom was prepared for the entire experiment in order to reduce variation among extractions.
Materials and methods
foenum graecum Hance; 7.1 g Citrus nobilis Lour.; 11.8 g Magnolia officinalis Rehd. et Wils; 7.1 g Cinnamonum cassia Presl (Chinese cinnamon); 3.5 g Mentha arvensis L.; 2.3 g Asarum sieboldii Mig. (Soie-cheng); 9.3 g Ligusticum wallichii Franch; 4.8 g Glycyrrhiza glabra L. (licorice); 7.1 g Eugenia caryophyllata Thunb. (clove); 7.1 g Saussurea lappa Clarke (Kote-kra-dook); 7.1 g Aquilaria agallocha Roxb.; 9.3 g Atraetlis ovata Thunb.; (Kote-kae-ma); 4.7 g menthol; 1.4 g Borneo camphor; and 3.5 g Angelica anomala Lallem (Kote-sor). Ya-hom powder was boiled in water in a ratio of 1 g: 20 ml for 15 min, filtered through cotton and muslin cloth, and the filtrate lyophilized and kept at 20 1C until use. One gram of Ya-hom powder yielded 0.136 g of lyophilized product. Ya-hom solution was freshly prepared on the day of experiment by redissolving the lyophilized powder in Kreb–Henseleit solution for aorta (KH aorta) and Kreb–Henseleit solution for heart (KH heart) to investigate the effect of Ya-hom on isolated aortic ring and isolated atrium, respectively. The concentrations of Ya-hom are expressed as concentrations of Ya-hom powder in the organ bath (mg/ml).
HPLC analysis of Ya-hom Lyophilized Ya-hom water extract was dissolved in methanol, sonicated for 20 min and then adjusted to a concentration of 0.01 g/ml with methanol. The sample was filtered through a Sep-packs C18 cartridge before applying to HPLC using LiChrospheres 100RP-18, Merck (5 mm) column and eluted with deionized water for 5 min, followed by a linear gradient of 10–100% acetonitrile for 15 min. The HPLC analysis of lyophilized Ya-hom water extract is shown in Fig. 1.
Animals Male Wistar rats weighing between 180–220 g each were obtained from the National Laboratory Animal Center at Salaya, Mahidol University, Nakornpratom, Thailand. The rats were housed in hanging cages in the animal room at the Faculty of Pharmacy, Mahidol University, and fed with commercial rat diet (FE Zeullic) and tap water ad libitum.
Ya-hom preparation Chemicals The selected brand of Ya-hom was purchased from the Ya-hom producing company, Bangkok, Thailand. A 100-g sample of Ya-hom contains: 7.1 g Agastache rugosa (Fisch. et Mey.) O. Kuntze; 3.5 g Acorus gramineus Soland (Wan-num-lek); 3.3 g Lysimachia
Norepinephrine (NE) bitartrate, phentolamine and propranolol were purchased from Sigma Chemical Co., St. Louis, USA. Other chemicals were of analytical grade.
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Fig. 1. HPLC analysis of lyophilized Ya-hom. Lyophilized Ya-hom in methanol was applied to HPLC using LiChrospheres 100RP-18, and a Merck (5 mm) column eluted with deionized water and a linear gradient of 10–100% acetonitrile.
Isolated aortic ring The thoracic aorta, between the aortic arch and diaphragm was removed from each ether-anesthesized rat and placed into the 37 1C-KH aorta solution, which was aerated continually with 95% O2 and 5% CO2. Kreb–Henseleit solution for the thoracic aorta was composed of 115 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mM KH2PO4 and 10 g/l of glucose; pH was adjusted to 7.4 with diluted HCl (Itoh and Lederis, 1987). The 7-mm aortic ring was fastened to a force displacement transducer, which was connected to a polygraph system recorder (Nihom Holden). The aortic ring was given force at 1 g. After 2 h equilibration, the aortic smooth muscle was in the resting length for optimal tension development when challenged with the maximum dose (105 M) of NE bitartrate (Hickey et al., 1985). The force of the contraction was recorded continuously until the maximum response was measured after adding each test solution. The effects of NE, Ya-hom and phentolamine were studied in the same aortic ring.
for 30 min. The effect of NE was recorded continuously until the maximum response was measured before washing with the KH heart solution. Because the response profile of Ya-hom was different from NE, the contraction of the atrium in response to Yahom was recorded at the first, second, third and fifth minute, and at 5-min intervals for 25 min after adding Ya-hom solution. The effect of propranolol on NE action was carried out in the same atrium but the investigation of the effects of Ya-hom and Ya-hom with propranolol were performed in a different atrium.
Statistical analysis One-way analysis of variance (ANOVA) was used to compare the values for more than two groups. The Duncan and Student–Newman–Keuls procedures were used to differentiate between the two experimental groups. Paired and unpaired Student’s t-tests were used to compare the values for the two groups. Statistical significance was measured as po0:05.
Isolated atrium
Results
The heart was removed from each ether-anesthesized rat. The atrium was isolated and placed into the 37 1CKH heart solution, which was aerated continuously with 95% O2 and 5% CO2. Kreb–Henseleit solution for the heart was composed of 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 24 mM NaHCO3, 1.2 mM KH2PO4 and 4.5 g/l of glucose; pH was adjusted to 7.4 with diluted HCl (Pery-Man et al., 1993). The rate and force of atrial contraction were measured by a force displacement transducer with an appropriate preamplifier and recorded on the chart paper of the polygraph system recorder. The atrium was equilibrated with 50 mg
Effects of Ya-hom on aortic ring contraction: the dose–response of Ya-hom and antagonist effect of the a-blocker NE, an endogenous vasoconstrictor, at the concentration of 1010–105 M increased aortic ring contraction concentration-dependently, with the maximum response at 106 M (Fig. 2). Phentolamine, an a-blocker, at the concentration of 10 nM, decreased the effect of 109–108 M NE significantly. Ya-hom at concentrations of 1.67, 8.33, 16.67 and 83.33 mg/ml induced aortic ring contraction with the maximum response at
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Fig. 2. Dose–response of NE, 1010–105 M, on aortic ring contraction in cumulative addition in the presence and absence of phentolamine 10 nM. Phentolamine was added 3 min before adding NE. All values were expressed as mean7s.e.m. (n ¼ 10). b (po0:005): significant difference from the NE alone, at corresponding concentration.
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Fig. 4. Effect of Ya-hom at the concentration of 1.67 mg/ml on force of aortic ring contraction, stimulated by NE at the concentration of 109 M, when given before, with and after NE. All values were expressed as mean7s.e.m. (n ¼ 10). The last column represented the force of aortic ring contraction when NE was added before Ya-hom in the presence of propranolol. There were no significant differences among four different conditions of adding NE, Ya-hom and propranolol. a (po0:05), b (po0:005): significant difference from NE alone.
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Fig. 3. Dose–response of Ya-hom at concentrations of 1.67, 8.33, 16.67 and 83.33 mg/ml on the aortic ring contraction, in cumulative addition, in the absence and presence of phentolamine 10, 20 and 40 nM. Phentolamine was added 3 min before adding Ya-hom. All values were expressed as mean7s.e.m. (n ¼ 10). a (po0:05), b (po0:005) : significant difference from Ya-hom alone at corresponding concentrations.
16.67 mg/ml (Fig. 3). Comparing the maximum effects of NE and Ya-hom showed that the potency of Ya-hom was about 14% of NE (110.6719.2 mg vs. 791.0762.0 mg). Phentolamine, at concentrations of 10, 20 and 40 nM, attenuated all four doses of Yahom-induced aortic ring contraction dose-dependently and significantly (Fig. 3).
Effect of Ya-hom on norepinephrine reponse Ya-hom (1.67 mg/ml) given 3 min before, with or after maximum response of NE caused significant decreases
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Fig. 5. Effect of NE 1010–104 M (cumulative addition) on the aortic ring in the absence and presence of Ya-hom at the concentration of 8.33 mg/ml. Ya-hom was added 3 min before adding NE. All values were expressed as mean7s.e.m. (n ¼ 10). b (po0:005): significant difference from the NE alone, at corresponding concentration.
in aortic ring contraction induced by NE 109 M from 116.3722.2 to 70.9714.6, 64.3711.2 and 62.8711.6, respectively (Fig. 4). The similar inhibitory effect of Yahom on NE response was observed independent of the sequence of addition. In the presence of propranolol, a b blocker, the inhibitory effect of Ya-hom on NE response
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was greater than in the absence of propranolol, but it was not significantly different. Ya-hom at the concentration of 8.33 mg/ml also shifted the dose–response curve of NE (1010–104 M) to the right (Fig. 5). This result indicated that Ya-hom itself increased aortic ring contraction; however, it decreased the stimulatory effect of NE when given together with it.
Effects of Ya-hom on isolated atrial contraction; effect of NE on the force and rate of atrial contraction
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Spontaneous contraction force and rate of atrial contraction prior to NE addition of each concentration were used as controls. In order to compare atrial response to NE with and without propranolol, the atrial 400 350 300 250
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Fig. 6. Log concentration–response curve of atrial contraction force induced by NE 108, 107, 106 and 105 M in the absence and presence of propranolol 106 M. All values were expressed as mean7s.e.m. (n ¼ 8). a (po0:05), b (po0:005): significant difference from NE alone.
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contraction force and rate were calculated as % spontaneous contraction. NE at concentrations of 108, 107, 106 and 105 M increased both the force and rate of atrial contraction. These stimulatory effects of NE were inhibited by a b blocker, propranolol, at 106 M (Figs. 6 and 7).
Effects of Ya-hom on the force and rate of atrial contraction Ya-hom at the concentration of 0.83 mg/ml increased the force of atrial contraction significantly at 1, 20 and 25 min (Fig. 8). Ya-hom at concentrations of 1.67 and 8.33 mg/ml increased atrial contraction force significantly over 30 min of observation, whereas Ya-hom at 16.67 mg/ml increased atrial contraction force significantly only from the first to the 20th minute (Fig. 8). To evaluate the effect of propranolol, atrial contraction force was calculated as % of spontaneous contraction. Propranolol (106 M) inhibited the force of atrial contraction significantly (% spontaneous contraction) increased by 1.67 mg/ml of Ya-hom at 2, 3, 4, 5 and 25 min (Fig. 9B). Propanolol, however, caused no significant change in the response of Ya-hom at the concentrations of 0.83, 8.33 and 16.67 mg/ml (Fig. 9A, 9C and 9D). The atrial contraction response profile to Ya-hom was different from those of NE. NE immediately increased force of atrial contraction with a short duration, while Ya-hom caused a slow increased force of contraction with a long duration. Moreover, the atrial contraction profile varied especially during the first 20 min when using different concentrations of Ya-hom. Time to
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Fig. 7. Log concentration–response curve of atrial contraction rate induced by NE 108, 107, 106 and 105 M in the absence and presence of propranolol 106 M. All values were expressed as mean of % spontaneous rate of atrial contraction7s.e.m. (n ¼ 8). a (po0:05), b (po0:005): significant difference from NE alone.
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Fig. 8. Effects of Ya-hom at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml on the force of atrial contractions at 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 min. c (po0:05), d (po0:005) : significant difference from the control (before adding Ya-hom).
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Fig. 9. Atrial contraction force (% spontaneous contraction) in response to Yahom (A) 0.83 mg/ml, (B) 1.67 mg/ml, (C) 8.33 mg/ml and (D) 16.67 mg/ml in the absence and presence of propranolol 106 M at 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 min. a (po0:05), b (po0:005): significant difference between the Ya-hom and Ya-hom+propranolol groups at corresponding times.
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Fig. 10. The maximum effect of Ya-hom at the concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml on atrial contraction in the absence and presence of propranolol 106 M. All values were expressed as % spontaneous contraction. There were no significant differences between the Ya-hom and Ya-hom+ propranolol group at corresponding concentration.
maximum response of the high concentration was prolonged with low concentrations (1, 5, 10 and 10 min at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml, respectively). The maximum response of atrial contraction to each concentration was therefore selected as another parameter for comparing the effects of Ya-hom. In terms of % spontaneous contraction, Yahom induced maximum force of atrial contraction in a
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Fig. 11. Effects of Ya-hom at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml on the rate of atrial contraction at 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 min. c (po0:05), d (po0:005) : significant difference from control.
concentration-dependent manner (Fig. 10). There were no significant differences between the maximum response of Ya-hom alone and Ya-hom with propranolol. These data indicated that Ya-hom had a stimulatory effect on the force of atrial contraction and most of this stimulation could not be inhibited by propranolol. Ya-hom at the concentrations of 0.83 and 1.67 mg/ml showed no effect on the rate of atrial contraction, whereas Ya-hom at concentrations of 8.33 and 16.67 mg/ml
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Fig. 12. Atrial contraction rate (% spontaneous contraction rate) in response to Yahom (A) 0.83 mg/ml, (B) 1.67 mg/ml, (C) 8.33 mg/ml and (D) 16.67 mg/ml in the absence and presence of propranolol 106 M at 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 min. There were no significant differences between the Ya-hom and Ya-hom+propranolol groups at corresponding times.
Discussion Effects of Ya-hom on aortic ring contraction The effects of Ya-hom on isolated rat aorta were investigated and compared to the actions of NE, an
120 Rate of atrial contraction (% spontaneous contraction)
decreased the rate of atrial contraction significantly (Fig. 11). Ya-hom at the concentration of 8.33 mg/ml decreased the rate of atrial contraction significantly, from 2 to 20 and 30 min, while at the concentration of 16.67 mg/ ml, depressed it significantly through 30 min (Fig. 11). Propranolol, which showed no effect on the positive inotropic action of Ya-hom, also had no influence on the negative chronotropic effect of Ya-hom (% spontaneous contraction rate) as shown in Fig. 12. The rate of atrial contraction decreased by Ya-hom was varied in the first 20 min after adding Ya-hom and stable after 20 min. The maximum response of the atrial contraction rate to each concentration was selected as another parameter for comparing the effect of Ya-hom. The atrial contraction rate was decreased significantly by Ya-hom only at concentrations of 8.33 and 16.67 mg/ ml. This negative chronotropic effect of Ya-hom was not altered by propranolol (Fig. 13).
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Fig. 13. The maximum effect of Ya-hom at the concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml in the absence and presence of propranolol 106 M. All values were expressed as % spontaneous atrial contraction rate. There were no significant differences between the Ya-hom and Ya-hom+propranolol groups at corresponding concentrations.
endogenous sympathetic neurotransmitter. NE (1010–105 M) increased aortic ring contraction in a concentration-dependent manner, with the maximum effect at the concentration of 106 M. NE acts on a1-receptor in the vascular smooth muscle cells, causing a vasoconstriction which is inhibited by phentolamine (a-blocking agent). In this study, phentolamine at the
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concentration of 10 nM inhibited the stimulatory effect of NE significantly only at the concentrations of 109 and 108 M. Ya-hom increased aortic ring contraction in a concentration-dependent manner, with the maximum response at 16.67 mg/ml. This stimulation was inhibited by phentolamine in a dose-dependent manner, from 10 to 40 nM. In addition, the concentration–force curve of Ya-hom in the presence of phentolamine was shifted to the right (Fig. 3). These results suggested that Ya-hom stimulated aortic ring contraction by acting on the a1-receptor of the vascular smooth muscle cells and its effect was blocked non-competitively by phentolamine. The mechanism of Ya-hom-induced aortic ring contraction was investigated further by studying the combined effect of Ya-hom and NE. It was found that adding Ya-hom, before, after or with NE, produced no significantly different response in aortic ring contraction. The responses to these combinations were lower than the effect of NE alone but higher than that of Yahom alone. Because the stimulatory effect of Ya-hom was inhibited by a1-antagonist and Ya-hom decreased the effect of a full agonist, NE, Ya-hom may act as a partial agonist on the a1-receptor. Ya-hom is a combination of medicinal plants. It is therefore possible that some ingredients of Ya-hom stimulate b-receptor, causing a relaxation of smooth muscle to a lesser extent than to the major stimulatory effect. The force of aortic ring contraction in the presence of propranolol was, however, not significantly lower than in the absence of propranolol. These data indicated that the inhibitory action of Ya-hom on the effect of NE did not depend on b-receptor stimulation. The present study shows that Ya-hom increases the force of aortic ring contraction by activating a1receptor, similar to NE, with lower potency. Ya-hom may act as partial a1-agonist in the aortic ring smooth muscle. Although the effect of Ya-hom on the isolated blood vessel has never been reported, and there are few reports about its effect on blood pressure, Ya-hom was shown, probably through a vasodilation effect, to decrease diastolic pressure and mean blood pressure in humans and rats (Matangkasombat, 1973; Wangmad et al., 1986). The water-soluble fraction of the chloroform and water extracts of Ya-hom, however, raised blood presssure in rats (Matangkasombat, 1974). The difference between the earlier reports and the present data may be due to: (a) the different methods of preparing Ya-hom, which result in different extracted ingredients; (b) the different experimental models (in vitro vs. in vivo), as the blood pressure in the in vivo study is regulated by several factors, whereas the isolated aortic contraction is affected directly by test solution; (c) the different brands of Ya-hom, containing different ingredients which can have different effects.
Effects of Ya-hom on atrial contraction In the positive control, NE showed both positive inotropic and positive chronotropic effects in a concentration-dependent manner. The force of the atrial contraction was maximal at 105 M, whereas the rate of contraction was maximal at 106 M with NE. These stimulatory effects of NE were inhibited by propranolol, a b-antagonist (106 M). The effect of Ya-hom was studied using a similar protocol to NE. Unlike the fast onset of NE action, Ya-hom had a slow onset and a long-lasting action which was similar to that reported by Na Pattaloong and Sawasdimongkol (1995). The atrium had to be washed with KH for heart solution several times in order to eliminate the effect of Ya-hom. Although propranolol inhibited the Ya-hom (1.67 mg/ ml)-increased force of atrial contraction significantly at some periods, propranolol did not alter the response of atrial contraction at other concentrations and the maximum response of every tested Ya-hom concentration. This indicated that the positive inotropic effect of Ya-hom is probably not related to b1-receptor stimulation. This conclusion is similar to that of Na Pattaloong and Sawasdimongkol (1995), who demonstrated that Ya-hom brand no. 1 increased the force but decreased the rate of isolated atrial contraction, whereas no. 4 only increased the force of isolated atrial contraction. Yahom brands no. 1 and 4 could decrease the negative inotropic effect of acetylcholine; they then proposed that the positive inotropic effect of Ya-hom might be due to the parasympatholytic effect (Na Pattaloong and Sawasdimongkol, 1995). In contrast to the stimulatory effect on the force of atrial contraction, Ya-hom decreased the rate of atrial contraction. Similar to the inotropic effect, the rate of atrial contraction in the presence of Ya-hom and propranolol did not differ significantly from that of Ya-hom alone, which showed the unrelated b1-receptor to the negative chronotropic effect of Ya-hom. This indicated that the negative chronotropic effect of Yahom was not involved in the adrenergic system. Matangkasombat (1973) also found that Ya-hom decreased the pulse rate in humans. Wangmad et al. (1986), however, showed that Ya-hom had no effect on rat heart rate. Na Pattaloong and Sawasdimongkol (1995), using the same brands of Ya-hom as Wangmad, demonstrated that Ya-hom brand no. 3 decreased the rate of isolated atrial contraction but did not have an effect on atrial contraction force. The effect of Ya-hom no. 3 could not antagonize the positive chronotropic effect of isoproterenol, which indicated that the negative chronotropic effect of Ya-hom was not a b-adrenergic antagonist (Na Pattaloong and Sawasdimongkol, 1995). The present study also showed that the negative chronotropic effect of Ya-hom did not involve the adrenergic receptor. The opposite effects of Ya-hom
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(positive inotropic and negative chronotropic) should be caused by the different mechanism and also different compositions. Although several formulas of Ya-hom have similar effects, the exact mechanism of both positive and negative chronotropic effects of each Yahom may be different and must be clarified further.
Conclusion The present study demonstrates that Ya-hom increases the force of isolated rat aortic ring contraction by stimulating the a-receptor on the vascular smooth muscle cells. It may act as a partial a-agonist. Ya-hom increases the force of isolated rat atrial contraction, with a slow onset and prolonged action and decreases the rate of atrial contraction, which does not involve the breceptor. The same four brands of Ya-hom studied in rats and isolated rat atrium showed contrasting results (Wangmad et al., 1986; Na Pattaloong and Sawasdimongkol, 1995). It will be interesting to further study the cardiovascular effects of this present brand of Yahom in vivo to demonstrate whether the in vivo effect is in correspondence with the in vitro study. Furthermore, this study provided a guide for studying the cardiovascular effects of other brands of Ya-hom.
Acknowledgement This research is partially supported by an M.S. research grant from the Faculty of Pharmacy, Mahidol University.
References Benbassat, J., Sulman, F.G., Zaitschek, D.V., 1959. Hypotensive effect of ligum aloes. Arch. Intern. Pharmacodynam. 120, 141–151. Bose, B.C., Saifi, A.Q., Vijayvargiya, R., Sharma, S.K., 1961. Some aspects of phytochemical and pharmaceutical study of Saussurea lappa Clarke. J. Pharm. Sci. 50 (8), 679–680. El-Mahdy, S.A., Ali, H.I., Helmi, R., Moustafa, M., 1973. Studies on the effect of Licorice on the circulation. J. Egypt Med. Assoc. 55 (11/12), 925–933.
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Fournier, A., Lagrue, G., 1971. Hypertension due to hypermineralocorticism. Sem. Hop. 47 (14), 872–889. Girerd, R.J., Rassaert, C.L., Di Pasquate, G., Kroc, R.L., 1958. Production of experimental hypertension and cardiovascular-renal lessions with licorice and ammonated glycyrrhzin. Am. J. Physiol. 194, 241–245. Gupta, O.P., Ghatak, B.J.R., 1967. Pharmacological investigations on Saussurea lappa (Clarke). Indian J. Med. Res. 55 (10), 1078–1083. Hano, K., Goto, T., Nakajima, I., 1950. Pharmacological properties of glycyrrhetic acid. Jpn. J. Pharm. Chem. 22, 325–334. Hickey, K., Rubanyi, G., Paul, R.J., Highsmith, R.F., 1985. Characterization of coronary vasocontrictor produced by cultured endothelial cells. Am. J. Physiol. 248, C550–C556. Itoh, H., Lederis, K., 1987. Contraction of rat thoracic aortic strips induced by phorbol 12-myristate 13-acetate. Am. J. Physiol. 252, C244–C247. Khadzhai, Y.I., Sokolova, V.E., 1960. Pharmacology of angesin, a crystalline compound from Angelica silvestris. Farmakol. Toksikol. 23 (1), 37–42. Kong, Y.C., Hu, S.Y., Lau, F.K., Che, C.T., Yeung, H.W., Cheung, S., Hwang, J.C.C., 1976. Potential anti-fertility plants from Chinese medicine. Am. J. Chinese Med. 4 (2), 105–128. Matangkasombat, O., 1973. Study on the cardiovascular effect of Ya-hom (a Thai folk medicine). Mahidol University Annual Research Abstract 67. Matangkasombat, O., 1974. Pharmacological effects of Thaifolk medicine (Ya-Hom) on the blood pressure and cardiac function in man and experimental animals. Complete Report submitted to the National Research Council of Thailand. Na Pattaloong, P., Sawasdimongkol, K., 1995. Action of Thai traditional cardiotonic preparations (Ya-Hom) on isolated rat atrium. Bull. Dept. Med. Sci. 37 (4), 271–288. Pery-Man, N., Chemla, D., Coirault, C., Suard, I., Riou, B., Lecarpentier, Y., 1993. A comparison of cyclopiazonic acid and ryanodine effects on the cardiac muscle relaxation. Am. J. Physiol. 265, H1364–H1372. Shihata, I.M., Elghamry, M.I., 1963. Experimental studies in the effect of Glycyrrhiza glabra. Planta Med. 11, 37–43. Shoji, N., Umeyama, A., Saito, N., Takemoto, T., 1986. Vasoactive substances from Saussurea lappa. J. Nat. Prod. 49 (6), 1112–1113. Wangmad, M., Anulakanapakorn, K., Sawasdimongkol, K., 1986. Cardiotonic property of traditional remedies (Ya-Hom). Bull. Dept. Med. Sci. 28 (4), 427–435.
Phytomedicine 12 (2005) 561–569 www.elsevier.de/phymed
Actions of Ya-hom, a herbal drug combination, on isolated rat aortic ring and atrial contractions W. Suvitayavata,, S. Tunlerta, S.S. Thirawarapana, C. Kitpatia, N. Bunyapraphatsarab a
Department of Physiology, Faculty of Pharmacy, Mahidol University, 447 Sri-ayuthaya Road, Bangkok 10400, Thailand Department of Phamacognosy, Faculty of Pharmacy, Mahidol University, 447 Sri-ayuthaya Road, Bangkok 10400, Thailand
b
Received 22 January 2004; accepted 5 March 2004
Abstract The effect of the Thai popular medicine Ya-hom on cardiovascular function was studied in isolated rat aortic ring and atrium by comparison with norepinephrine (NE). Water extraction of Ya-hom at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml stimulated aortic ring contraction dose-dependently. The maximum contraction, at 16.67 mg/ml, was about 14% that of NE. This stimulatory effect of Ya-hom was inhibited partially by phentolamine, which indicated that the effect of Ya-hom was partially dependent on the a receptor, similar to NE. Administration of Ya-hom with NR decreased the force of aortic ring contraction as compared to the effect of NE alone, indicating that Ya-hom may have a partial a-agonist activity. Ya-hom at concentrations of 1.67, 8.33 and 16.67 mg/ml showed a dose-dependent, positive inotropic and negative chronotropic effects. Ya-hom increased the force of isolated atrial contraction with a slow onset and prolonged action. In contrast to norephinephrine, which acted on b1 receptor, causing positive inotropic and chronotropic effects, propranolol did not alter the effect of Ya-hom on the atrial contraction. This shows that the action of Ya-hom on atrial contraction does not involve b receptor. This study demonstrated that the selected Ya-hom preparation increased vascular smooth muscle contraction, and increased the force but decreased the rate of atrial contraction. r 2005 Elsevier GmbH. All rights reserved. Keywords: Ya-hom; Aortic ring; Rat atrium; Cardiovascular effects
Introduction Ya-hom, one of the most popular Thai folk medicines, has been used for treatment of fainting, nausea and vomiting. Ya-hom preparations are marketed under different trade names with different compositions as well as different proportion of medicinal plants. The Corresponding author.
E-mail address: [email protected] (W. Suvitayavat). 0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.03.014
cardiovascular effects of some principal ingredients in Ya-hom have been reported. Aquilaria agallocha Roxb (Benbassat et al., 1959), Kote-sor (Khadzhai and Sokolova, 1960) and Chinese cinnamon (Kong et al., 1976) have been reported to cause vasodilation and decrease blood pressure, whereas aqueous and alcoholic extracts of Kote-kra-dook caused an increase in blood pressure (Bose et al., 1961). The methanol extract of Kote-kra-dook, however, inhibited KCl-induced aortic contraction (Shoji et al., 1986) and was also shown to
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have a hypotensive action (Gupta and Ghatak, 1967). Reports showed that licorice decreased (El-Mahdy et al., 1973; Shihata and Elghamry, 1963) and increased blood pressure (Hano et al., 1950; Fournier and Lagrue, 1971; Girerd et al., 1958), stimulated and depressed myocardium function and caused vasoconstriction and vasodilation according to concentration (El-Mahdy et al., 1973). Most of the Ya-hom recipes are composed of both cardiovascular stimulatory and inhibitory herbal medicines. Therefore, the exact action of each formula must be investigated. There have been few reports about the effect of Yahom on both animal and human cardiovascular function. Ya-hom slowed the pulse rate and widened the pulse pressure in humans (Matangkasombat, 1973). In the rat, the water-soluble fraction of a chloroform extract and a water extract of Ya-hom extracts raised blood pressure, whereas the alcohol extract and waterinsoluble fraction of the chloroform extract lowered blood pressure (Matangkasombat, 1974). In addition, Ya-hom extracts have a direct stimulating effect on the rat heart, whereas they show no effect on the rabbit heart (Matangkasombat, 1974). Another report using water extraction indicated that four brands of Ya-hom decreased blood pressure but had no effect on the heart rate, pulse pressure or electrocardiogram of rats (Wangmad et al., 1986). Two of these four brands (nos. 1 and 4) had a positive inotropic effect, two of them (nos. 1 and 3) had negative chronotropic effect, whereas the remaining one (no. 2) had no effect on the isolated atrium (Na Pattaloong and Sawasdimongkol, 1995). According to some controversial results, determination of the effect of Ya-hom on the cardiovascular function remains inconclusive. This may be due to use of the different brands, extraction methods and experimental models. Because most Ya-hom formulae contain similar major ingredients, the most famous brand of Yahom has been selected for the study of its cardiac and vascular effects, in vitro in this present study and using an in vivo model for the next study. The pharmacological actions of this brand have never been reported. A massive single preparation of a lyophilized water extract of Ya-hom was prepared for the entire experiment in order to reduce variation among extractions.
Materials and methods
foenum graecum Hance; 7.1 g Citrus nobilis Lour.; 11.8 g Magnolia officinalis Rehd. et Wils; 7.1 g Cinnamonum cassia Presl (Chinese cinnamon); 3.5 g Mentha arvensis L.; 2.3 g Asarum sieboldii Mig. (Soie-cheng); 9.3 g Ligusticum wallichii Franch; 4.8 g Glycyrrhiza glabra L. (licorice); 7.1 g Eugenia caryophyllata Thunb. (clove); 7.1 g Saussurea lappa Clarke (Kote-kra-dook); 7.1 g Aquilaria agallocha Roxb.; 9.3 g Atraetlis ovata Thunb.; (Kote-kae-ma); 4.7 g menthol; 1.4 g Borneo camphor; and 3.5 g Angelica anomala Lallem (Kote-sor). Ya-hom powder was boiled in water in a ratio of 1 g: 20 ml for 15 min, filtered through cotton and muslin cloth, and the filtrate lyophilized and kept at 20 1C until use. One gram of Ya-hom powder yielded 0.136 g of lyophilized product. Ya-hom solution was freshly prepared on the day of experiment by redissolving the lyophilized powder in Kreb–Henseleit solution for aorta (KH aorta) and Kreb–Henseleit solution for heart (KH heart) to investigate the effect of Ya-hom on isolated aortic ring and isolated atrium, respectively. The concentrations of Ya-hom are expressed as concentrations of Ya-hom powder in the organ bath (mg/ml).
HPLC analysis of Ya-hom Lyophilized Ya-hom water extract was dissolved in methanol, sonicated for 20 min and then adjusted to a concentration of 0.01 g/ml with methanol. The sample was filtered through a Sep-packs C18 cartridge before applying to HPLC using LiChrospheres 100RP-18, Merck (5 mm) column and eluted with deionized water for 5 min, followed by a linear gradient of 10–100% acetonitrile for 15 min. The HPLC analysis of lyophilized Ya-hom water extract is shown in Fig. 1.
Animals Male Wistar rats weighing between 180–220 g each were obtained from the National Laboratory Animal Center at Salaya, Mahidol University, Nakornpratom, Thailand. The rats were housed in hanging cages in the animal room at the Faculty of Pharmacy, Mahidol University, and fed with commercial rat diet (FE Zeullic) and tap water ad libitum.
Ya-hom preparation Chemicals The selected brand of Ya-hom was purchased from the Ya-hom producing company, Bangkok, Thailand. A 100-g sample of Ya-hom contains: 7.1 g Agastache rugosa (Fisch. et Mey.) O. Kuntze; 3.5 g Acorus gramineus Soland (Wan-num-lek); 3.3 g Lysimachia
Norepinephrine (NE) bitartrate, phentolamine and propranolol were purchased from Sigma Chemical Co., St. Louis, USA. Other chemicals were of analytical grade.
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Fig. 1. HPLC analysis of lyophilized Ya-hom. Lyophilized Ya-hom in methanol was applied to HPLC using LiChrospheres 100RP-18, and a Merck (5 mm) column eluted with deionized water and a linear gradient of 10–100% acetonitrile.
Isolated aortic ring The thoracic aorta, between the aortic arch and diaphragm was removed from each ether-anesthesized rat and placed into the 37 1C-KH aorta solution, which was aerated continually with 95% O2 and 5% CO2. Kreb–Henseleit solution for the thoracic aorta was composed of 115 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mM KH2PO4 and 10 g/l of glucose; pH was adjusted to 7.4 with diluted HCl (Itoh and Lederis, 1987). The 7-mm aortic ring was fastened to a force displacement transducer, which was connected to a polygraph system recorder (Nihom Holden). The aortic ring was given force at 1 g. After 2 h equilibration, the aortic smooth muscle was in the resting length for optimal tension development when challenged with the maximum dose (105 M) of NE bitartrate (Hickey et al., 1985). The force of the contraction was recorded continuously until the maximum response was measured after adding each test solution. The effects of NE, Ya-hom and phentolamine were studied in the same aortic ring.
for 30 min. The effect of NE was recorded continuously until the maximum response was measured before washing with the KH heart solution. Because the response profile of Ya-hom was different from NE, the contraction of the atrium in response to Yahom was recorded at the first, second, third and fifth minute, and at 5-min intervals for 25 min after adding Ya-hom solution. The effect of propranolol on NE action was carried out in the same atrium but the investigation of the effects of Ya-hom and Ya-hom with propranolol were performed in a different atrium.
Statistical analysis One-way analysis of variance (ANOVA) was used to compare the values for more than two groups. The Duncan and Student–Newman–Keuls procedures were used to differentiate between the two experimental groups. Paired and unpaired Student’s t-tests were used to compare the values for the two groups. Statistical significance was measured as po0:05.
Isolated atrium
Results
The heart was removed from each ether-anesthesized rat. The atrium was isolated and placed into the 37 1CKH heart solution, which was aerated continuously with 95% O2 and 5% CO2. Kreb–Henseleit solution for the heart was composed of 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 24 mM NaHCO3, 1.2 mM KH2PO4 and 4.5 g/l of glucose; pH was adjusted to 7.4 with diluted HCl (Pery-Man et al., 1993). The rate and force of atrial contraction were measured by a force displacement transducer with an appropriate preamplifier and recorded on the chart paper of the polygraph system recorder. The atrium was equilibrated with 50 mg
Effects of Ya-hom on aortic ring contraction: the dose–response of Ya-hom and antagonist effect of the a-blocker NE, an endogenous vasoconstrictor, at the concentration of 1010–105 M increased aortic ring contraction concentration-dependently, with the maximum response at 106 M (Fig. 2). Phentolamine, an a-blocker, at the concentration of 10 nM, decreased the effect of 109–108 M NE significantly. Ya-hom at concentrations of 1.67, 8.33, 16.67 and 83.33 mg/ml induced aortic ring contraction with the maximum response at
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Fig. 4. Effect of Ya-hom at the concentration of 1.67 mg/ml on force of aortic ring contraction, stimulated by NE at the concentration of 109 M, when given before, with and after NE. All values were expressed as mean7s.e.m. (n ¼ 10). The last column represented the force of aortic ring contraction when NE was added before Ya-hom in the presence of propranolol. There were no significant differences among four different conditions of adding NE, Ya-hom and propranolol. a (po0:05), b (po0:005): significant difference from NE alone.
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Fig. 3. Dose–response of Ya-hom at concentrations of 1.67, 8.33, 16.67 and 83.33 mg/ml on the aortic ring contraction, in cumulative addition, in the absence and presence of phentolamine 10, 20 and 40 nM. Phentolamine was added 3 min before adding Ya-hom. All values were expressed as mean7s.e.m. (n ¼ 10). a (po0:05), b (po0:005) : significant difference from Ya-hom alone at corresponding concentrations.
16.67 mg/ml (Fig. 3). Comparing the maximum effects of NE and Ya-hom showed that the potency of Ya-hom was about 14% of NE (110.6719.2 mg vs. 791.0762.0 mg). Phentolamine, at concentrations of 10, 20 and 40 nM, attenuated all four doses of Yahom-induced aortic ring contraction dose-dependently and significantly (Fig. 3).
Effect of Ya-hom on norepinephrine reponse Ya-hom (1.67 mg/ml) given 3 min before, with or after maximum response of NE caused significant decreases
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Fig. 5. Effect of NE 1010–104 M (cumulative addition) on the aortic ring in the absence and presence of Ya-hom at the concentration of 8.33 mg/ml. Ya-hom was added 3 min before adding NE. All values were expressed as mean7s.e.m. (n ¼ 10). b (po0:005): significant difference from the NE alone, at corresponding concentration.
in aortic ring contraction induced by NE 109 M from 116.3722.2 to 70.9714.6, 64.3711.2 and 62.8711.6, respectively (Fig. 4). The similar inhibitory effect of Yahom on NE response was observed independent of the sequence of addition. In the presence of propranolol, a b blocker, the inhibitory effect of Ya-hom on NE response
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was greater than in the absence of propranolol, but it was not significantly different. Ya-hom at the concentration of 8.33 mg/ml also shifted the dose–response curve of NE (1010–104 M) to the right (Fig. 5). This result indicated that Ya-hom itself increased aortic ring contraction; however, it decreased the stimulatory effect of NE when given together with it.
Effects of Ya-hom on isolated atrial contraction; effect of NE on the force and rate of atrial contraction
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contraction force and rate were calculated as % spontaneous contraction. NE at concentrations of 108, 107, 106 and 105 M increased both the force and rate of atrial contraction. These stimulatory effects of NE were inhibited by a b blocker, propranolol, at 106 M (Figs. 6 and 7).
Effects of Ya-hom on the force and rate of atrial contraction Ya-hom at the concentration of 0.83 mg/ml increased the force of atrial contraction significantly at 1, 20 and 25 min (Fig. 8). Ya-hom at concentrations of 1.67 and 8.33 mg/ml increased atrial contraction force significantly over 30 min of observation, whereas Ya-hom at 16.67 mg/ml increased atrial contraction force significantly only from the first to the 20th minute (Fig. 8). To evaluate the effect of propranolol, atrial contraction force was calculated as % of spontaneous contraction. Propranolol (106 M) inhibited the force of atrial contraction significantly (% spontaneous contraction) increased by 1.67 mg/ml of Ya-hom at 2, 3, 4, 5 and 25 min (Fig. 9B). Propanolol, however, caused no significant change in the response of Ya-hom at the concentrations of 0.83, 8.33 and 16.67 mg/ml (Fig. 9A, 9C and 9D). The atrial contraction response profile to Ya-hom was different from those of NE. NE immediately increased force of atrial contraction with a short duration, while Ya-hom caused a slow increased force of contraction with a long duration. Moreover, the atrial contraction profile varied especially during the first 20 min when using different concentrations of Ya-hom. Time to
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Fig. 10. The maximum effect of Ya-hom at the concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml on atrial contraction in the absence and presence of propranolol 106 M. All values were expressed as % spontaneous contraction. There were no significant differences between the Ya-hom and Ya-hom+ propranolol group at corresponding concentration.
maximum response of the high concentration was prolonged with low concentrations (1, 5, 10 and 10 min at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml, respectively). The maximum response of atrial contraction to each concentration was therefore selected as another parameter for comparing the effects of Ya-hom. In terms of % spontaneous contraction, Yahom induced maximum force of atrial contraction in a
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Fig. 11. Effects of Ya-hom at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml on the rate of atrial contraction at 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 min. c (po0:05), d (po0:005) : significant difference from control.
concentration-dependent manner (Fig. 10). There were no significant differences between the maximum response of Ya-hom alone and Ya-hom with propranolol. These data indicated that Ya-hom had a stimulatory effect on the force of atrial contraction and most of this stimulation could not be inhibited by propranolol. Ya-hom at the concentrations of 0.83 and 1.67 mg/ml showed no effect on the rate of atrial contraction, whereas Ya-hom at concentrations of 8.33 and 16.67 mg/ml
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Fig. 12. Atrial contraction rate (% spontaneous contraction rate) in response to Yahom (A) 0.83 mg/ml, (B) 1.67 mg/ml, (C) 8.33 mg/ml and (D) 16.67 mg/ml in the absence and presence of propranolol 106 M at 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 min. There were no significant differences between the Ya-hom and Ya-hom+propranolol groups at corresponding times.
Discussion Effects of Ya-hom on aortic ring contraction The effects of Ya-hom on isolated rat aorta were investigated and compared to the actions of NE, an
120 Rate of atrial contraction (% spontaneous contraction)
decreased the rate of atrial contraction significantly (Fig. 11). Ya-hom at the concentration of 8.33 mg/ml decreased the rate of atrial contraction significantly, from 2 to 20 and 30 min, while at the concentration of 16.67 mg/ ml, depressed it significantly through 30 min (Fig. 11). Propranolol, which showed no effect on the positive inotropic action of Ya-hom, also had no influence on the negative chronotropic effect of Ya-hom (% spontaneous contraction rate) as shown in Fig. 12. The rate of atrial contraction decreased by Ya-hom was varied in the first 20 min after adding Ya-hom and stable after 20 min. The maximum response of the atrial contraction rate to each concentration was selected as another parameter for comparing the effect of Ya-hom. The atrial contraction rate was decreased significantly by Ya-hom only at concentrations of 8.33 and 16.67 mg/ ml. This negative chronotropic effect of Ya-hom was not altered by propranolol (Fig. 13).
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Fig. 13. The maximum effect of Ya-hom at the concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml in the absence and presence of propranolol 106 M. All values were expressed as % spontaneous atrial contraction rate. There were no significant differences between the Ya-hom and Ya-hom+propranolol groups at corresponding concentrations.
endogenous sympathetic neurotransmitter. NE (1010–105 M) increased aortic ring contraction in a concentration-dependent manner, with the maximum effect at the concentration of 106 M. NE acts on a1-receptor in the vascular smooth muscle cells, causing a vasoconstriction which is inhibited by phentolamine (a-blocking agent). In this study, phentolamine at the
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concentration of 10 nM inhibited the stimulatory effect of NE significantly only at the concentrations of 109 and 108 M. Ya-hom increased aortic ring contraction in a concentration-dependent manner, with the maximum response at 16.67 mg/ml. This stimulation was inhibited by phentolamine in a dose-dependent manner, from 10 to 40 nM. In addition, the concentration–force curve of Ya-hom in the presence of phentolamine was shifted to the right (Fig. 3). These results suggested that Ya-hom stimulated aortic ring contraction by acting on the a1-receptor of the vascular smooth muscle cells and its effect was blocked non-competitively by phentolamine. The mechanism of Ya-hom-induced aortic ring contraction was investigated further by studying the combined effect of Ya-hom and NE. It was found that adding Ya-hom, before, after or with NE, produced no significantly different response in aortic ring contraction. The responses to these combinations were lower than the effect of NE alone but higher than that of Yahom alone. Because the stimulatory effect of Ya-hom was inhibited by a1-antagonist and Ya-hom decreased the effect of a full agonist, NE, Ya-hom may act as a partial agonist on the a1-receptor. Ya-hom is a combination of medicinal plants. It is therefore possible that some ingredients of Ya-hom stimulate b-receptor, causing a relaxation of smooth muscle to a lesser extent than to the major stimulatory effect. The force of aortic ring contraction in the presence of propranolol was, however, not significantly lower than in the absence of propranolol. These data indicated that the inhibitory action of Ya-hom on the effect of NE did not depend on b-receptor stimulation. The present study shows that Ya-hom increases the force of aortic ring contraction by activating a1receptor, similar to NE, with lower potency. Ya-hom may act as partial a1-agonist in the aortic ring smooth muscle. Although the effect of Ya-hom on the isolated blood vessel has never been reported, and there are few reports about its effect on blood pressure, Ya-hom was shown, probably through a vasodilation effect, to decrease diastolic pressure and mean blood pressure in humans and rats (Matangkasombat, 1973; Wangmad et al., 1986). The water-soluble fraction of the chloroform and water extracts of Ya-hom, however, raised blood presssure in rats (Matangkasombat, 1974). The difference between the earlier reports and the present data may be due to: (a) the different methods of preparing Ya-hom, which result in different extracted ingredients; (b) the different experimental models (in vitro vs. in vivo), as the blood pressure in the in vivo study is regulated by several factors, whereas the isolated aortic contraction is affected directly by test solution; (c) the different brands of Ya-hom, containing different ingredients which can have different effects.
Effects of Ya-hom on atrial contraction In the positive control, NE showed both positive inotropic and positive chronotropic effects in a concentration-dependent manner. The force of the atrial contraction was maximal at 105 M, whereas the rate of contraction was maximal at 106 M with NE. These stimulatory effects of NE were inhibited by propranolol, a b-antagonist (106 M). The effect of Ya-hom was studied using a similar protocol to NE. Unlike the fast onset of NE action, Ya-hom had a slow onset and a long-lasting action which was similar to that reported by Na Pattaloong and Sawasdimongkol (1995). The atrium had to be washed with KH for heart solution several times in order to eliminate the effect of Ya-hom. Although propranolol inhibited the Ya-hom (1.67 mg/ ml)-increased force of atrial contraction significantly at some periods, propranolol did not alter the response of atrial contraction at other concentrations and the maximum response of every tested Ya-hom concentration. This indicated that the positive inotropic effect of Ya-hom is probably not related to b1-receptor stimulation. This conclusion is similar to that of Na Pattaloong and Sawasdimongkol (1995), who demonstrated that Ya-hom brand no. 1 increased the force but decreased the rate of isolated atrial contraction, whereas no. 4 only increased the force of isolated atrial contraction. Yahom brands no. 1 and 4 could decrease the negative inotropic effect of acetylcholine; they then proposed that the positive inotropic effect of Ya-hom might be due to the parasympatholytic effect (Na Pattaloong and Sawasdimongkol, 1995). In contrast to the stimulatory effect on the force of atrial contraction, Ya-hom decreased the rate of atrial contraction. Similar to the inotropic effect, the rate of atrial contraction in the presence of Ya-hom and propranolol did not differ significantly from that of Ya-hom alone, which showed the unrelated b1-receptor to the negative chronotropic effect of Ya-hom. This indicated that the negative chronotropic effect of Yahom was not involved in the adrenergic system. Matangkasombat (1973) also found that Ya-hom decreased the pulse rate in humans. Wangmad et al. (1986), however, showed that Ya-hom had no effect on rat heart rate. Na Pattaloong and Sawasdimongkol (1995), using the same brands of Ya-hom as Wangmad, demonstrated that Ya-hom brand no. 3 decreased the rate of isolated atrial contraction but did not have an effect on atrial contraction force. The effect of Ya-hom no. 3 could not antagonize the positive chronotropic effect of isoproterenol, which indicated that the negative chronotropic effect of Ya-hom was not a b-adrenergic antagonist (Na Pattaloong and Sawasdimongkol, 1995). The present study also showed that the negative chronotropic effect of Ya-hom did not involve the adrenergic receptor. The opposite effects of Ya-hom
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(positive inotropic and negative chronotropic) should be caused by the different mechanism and also different compositions. Although several formulas of Ya-hom have similar effects, the exact mechanism of both positive and negative chronotropic effects of each Yahom may be different and must be clarified further.
Conclusion The present study demonstrates that Ya-hom increases the force of isolated rat aortic ring contraction by stimulating the a-receptor on the vascular smooth muscle cells. It may act as a partial a-agonist. Ya-hom increases the force of isolated rat atrial contraction, with a slow onset and prolonged action and decreases the rate of atrial contraction, which does not involve the breceptor. The same four brands of Ya-hom studied in rats and isolated rat atrium showed contrasting results (Wangmad et al., 1986; Na Pattaloong and Sawasdimongkol, 1995). It will be interesting to further study the cardiovascular effects of this present brand of Yahom in vivo to demonstrate whether the in vivo effect is in correspondence with the in vitro study. Furthermore, this study provided a guide for studying the cardiovascular effects of other brands of Ya-hom.
Acknowledgement This research is partially supported by an M.S. research grant from the Faculty of Pharmacy, Mahidol University.
References Benbassat, J., Sulman, F.G., Zaitschek, D.V., 1959. Hypotensive effect of ligum aloes. Arch. Intern. Pharmacodynam. 120, 141–151. Bose, B.C., Saifi, A.Q., Vijayvargiya, R., Sharma, S.K., 1961. Some aspects of phytochemical and pharmaceutical study of Saussurea lappa Clarke. J. Pharm. Sci. 50 (8), 679–680. El-Mahdy, S.A., Ali, H.I., Helmi, R., Moustafa, M., 1973. Studies on the effect of Licorice on the circulation. J. Egypt Med. Assoc. 55 (11/12), 925–933.
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Fournier, A., Lagrue, G., 1971. Hypertension due to hypermineralocorticism. Sem. Hop. 47 (14), 872–889. Girerd, R.J., Rassaert, C.L., Di Pasquate, G., Kroc, R.L., 1958. Production of experimental hypertension and cardiovascular-renal lessions with licorice and ammonated glycyrrhzin. Am. J. Physiol. 194, 241–245. Gupta, O.P., Ghatak, B.J.R., 1967. Pharmacological investigations on Saussurea lappa (Clarke). Indian J. Med. Res. 55 (10), 1078–1083. Hano, K., Goto, T., Nakajima, I., 1950. Pharmacological properties of glycyrrhetic acid. Jpn. J. Pharm. Chem. 22, 325–334. Hickey, K., Rubanyi, G., Paul, R.J., Highsmith, R.F., 1985. Characterization of coronary vasocontrictor produced by cultured endothelial cells. Am. J. Physiol. 248, C550–C556. Itoh, H., Lederis, K., 1987. Contraction of rat thoracic aortic strips induced by phorbol 12-myristate 13-acetate. Am. J. Physiol. 252, C244–C247. Khadzhai, Y.I., Sokolova, V.E., 1960. Pharmacology of angesin, a crystalline compound from Angelica silvestris. Farmakol. Toksikol. 23 (1), 37–42. Kong, Y.C., Hu, S.Y., Lau, F.K., Che, C.T., Yeung, H.W., Cheung, S., Hwang, J.C.C., 1976. Potential anti-fertility plants from Chinese medicine. Am. J. Chinese Med. 4 (2), 105–128. Matangkasombat, O., 1973. Study on the cardiovascular effect of Ya-hom (a Thai folk medicine). Mahidol University Annual Research Abstract 67. Matangkasombat, O., 1974. Pharmacological effects of Thaifolk medicine (Ya-Hom) on the blood pressure and cardiac function in man and experimental animals. Complete Report submitted to the National Research Council of Thailand. Na Pattaloong, P., Sawasdimongkol, K., 1995. Action of Thai traditional cardiotonic preparations (Ya-Hom) on isolated rat atrium. Bull. Dept. Med. Sci. 37 (4), 271–288. Pery-Man, N., Chemla, D., Coirault, C., Suard, I., Riou, B., Lecarpentier, Y., 1993. A comparison of cyclopiazonic acid and ryanodine effects on the cardiac muscle relaxation. Am. J. Physiol. 265, H1364–H1372. Shihata, I.M., Elghamry, M.I., 1963. Experimental studies in the effect of Glycyrrhiza glabra. Planta Med. 11, 37–43. Shoji, N., Umeyama, A., Saito, N., Takemoto, T., 1986. Vasoactive substances from Saussurea lappa. J. Nat. Prod. 49 (6), 1112–1113. Wangmad, M., Anulakanapakorn, K., Sawasdimongkol, K., 1986. Cardiotonic property of traditional remedies (Ya-Hom). Bull. Dept. Med. Sci. 28 (4), 427–435.